An Introduction to Molecular Medicine and Gene Therapy Edited by Thomas F Kresina, PhD Copyright © 2001 by Wiley-Liss, Inc ISBNs: 0-471-39188-3 (Hardback); 0-471-22387-5 (Electronic) CHAPTER Gene Therapy for Hematological Disorders CYNTHIA E DUNBAR, M.D and TONG WU, M.D INTRODUCTION Hematopoietic cells are an attractive target for gene therapy for two main reasons First, it is possible to easily collect and then manipulate hematopoietic cells in vitro Second, many congenital and acquired diseases are potentially curable by genetic correction of hematopoietic cells, especially hematopoietic stem cells (HSCs, see Fig 6.1) For hematological disorders, the target cell(s) in which gene expression is required are red blood cells (RBC), lymphocytes, granulocytes, or other mature blood elements Ideally, the transgene is integrated into the chromatin of pluripotent HSCs, ensuring the continuous production of genetically modified blood cells of the desired lineage for the lifetime of the patient Other potential cellular targets with potential utility in the treatment of hematologic diseases include dendritic cells, tumor cells, and endothelial cells Hepatocytes, myocytes, and keratinocytes can be considered as “factories” for soluble factors with clinical utility in hematologic diseases such as hemophilia (see Chapter 7) Relevant targets and applications for gene therapy of hematopoietic or immune system disorders are summarized in Table 6.1 Many important advances in our understanding of hematopoiesis, stem cell engraftment, and other basic principles have resulted from animal models, in vitro studies, and early clinical trials of gene marking or gene therapy For example, studies using retrovirally marked murine stem cells show tracking and a quantitative analysis of murine stem cell behavior Experiments overexpressing oncogenes or cytokines in hematopoietic cells have elucidated the in vivo role of these proteins Early clinical gene marking trials demonstrated the long-term engrafting capability of peripheral blood stem cells The observed lack of clinical utility results from several major hurdles, including inefficient gene transfer to desired target cells, especially stem cells, poor in vivo expression of introduced genes, and immune responses against gene products recognized as foreign Further basic research investigations 133 134 GENE THERAPY FOR HEMATOLOGICAL DISORDERS FIGURE 6.1 Hierachal model of lymphohemopoiesis A primitive lymphohemopoietic cell is capable of producing lymphoid stem cells for lymphopoiesis or myeloid stem cells for hemopoiesis These stem cells give rise to progressively more differentiated progenitor cells that eventually give rise to lineage-specific terminally differentiated effector cells into new or modified vector systems and target cell biology are necessary to move the field forward into real clinical utility REQUIREMENTS FOR GENE TRANSFER INTO HEMATOPOIETIC CELLS Ex Vivo Versus in Vivo Gene Transfer Specific aspects of gene transfer techniques are advantageous for gene therapy approaches when applied to hematological diseases Aspects of ex vivo gene transfer as well as certain gene transfer vector systems are particularly useful in the experimental therapy of hematological diseases Hematopoietic cells such as stem cells or lymphocytes are generally transduced ex vivo because these cells can be easily collected, cultured, and transduced in vitro (see Chapter 1) Subsequently, they can be reinfused intravenously Ex vivo transduction allows for a controlled exposure of only the desired targets to vector particles It is less likely to produce an immune response or be impeded by complement-induced vector inactivation However, limited data indicate that direct in vivo injection of vector into the marrow space can transduce primitive cells But, there is no evidence that this in vivo method currently has any advantages over the more fully characterized ex vivo transduction approaches In vivo gene transfer is most appropriate for target cells that cannot REQUIREMENTS FOR GENE TRANSFER INTO HEMATOPOIETIC CELLS 135 TABLE 6.1 Relevant Targets and Applications for Gene Therapy of Hematopoietic or Immune System Disorders Target Cell or Lineage Example of Clinical Application Hematopoietic stem cells Fanconi anemia Red blood cells Thalassemia, sickle cell anemia Granulocytes Chronic granulomatous disease Lymphocytes Immunodeficiency diseases Cancer (TIL) AIDS (intracellular immunization) Macrophage Gaucher disease Dendritic cells Immune therapy Tumor cells Tumor suppressing genes Antisense to oncogenes Tumor vaccines Suicide genes Endothelial cells Inhibitors of thrombosis Growth factors Hepatocytes, myocytes Keratinocytes Hemophilia be easily harvested or manipulated ex vivo, such as airway epithelium, vascular endothelium, and differentiated muscle cells Vector Systems and Nonviral Vectors The choice of an appropriate vector system depends on the biology of the desired target cell and the need for transient versus prolonged gene expresssion (see Chapter 4) Both viral and nonviral vectors have been utilized to transduce hematopoietic target cells If prolonged correction or modification of hematopoietic cells is required, then vectors such as retroviruses that efficiently integrate into target cell chromosomes are necessary, otherwise new genetic material will be lost as HSCs or other targets such as lymphocytes proliferate On the other hand, if transient expression is required, for instance, in the production of leukemic cell tumor vaccines, then nonintegrating but efficiently expressing vectors such as adenoviruses may be preferred The vast majority of preclinical and clinical investigations of hematopoietic cell gene transfer utilize viral vectors, taking advantage of the characteristics of the virus that have evolved over time to efficiently infect target cells The viral genes and replication machinery are replaced with nonviral transgene sequences of interest For murine retroviruses, the Moloney murine leukemia virus (MuLV) vectors are the vectors of choice since they have not been supplanted by any other vector system for most hematologic applications Thus, MuLV vectors have been employed in almost every clinical study to date The main advantages of MuLV vectors are their ability to integrate a stable proviral form into the target cell genome, the availability of stable producer cell lines, the lack of toxicity to target cells, and almost 10 years of experience in using them safely in clinical trials Over the past several years, 136 GENE THERAPY FOR HEMATOLOGICAL DISORDERS FIGURE 6.2 Importance of cellular activation by growth factors or cytokines to induce mitosis for transduction by Moloney murine leukemia virus (MuLV) Cells must pass through the mitotic phase of the cell cycle (M, middle frame) in order for the vector to gain access to the chromatin and integrate into the genome (right frame) a number of modifications in the genetic sequences included in packaging cell lines has greatly decreased the risk of recombination events, and sensitive methods for detecting replication-competent virus have been established and are strictly utilized in all clinical trials There have been no documented adverse events related to insertional mutagenesis in early human clinical studies or in preclinical animal studies using replication-defective viral vectors There appear to be two major limitations to the use of MuLV vectors for hematopoietic stem cell transduction First, cells must pass through the mitotic phase of the cell cycle in order for the vector to gain access to the chromatin and integrate (Fig 6.2) Most stem cells reside in the G0 phase of the cell cycle, and manipulations that stimulate these cells to cycle ex vivo may result in irreversible lineage commitment or apoptosis Second, the receptor for MuLV retroviral vectors (amphotropic vectors) on human and primate cells has been identified and appears to be broadly expressed in most human tissues However, the low levels of this receptor on primitive HSCs may be limiting To redirect receptor specificity, pseudotyping of vectors has been employed by replacement of MuLV envelope proteins with gibbon ape leukemia virus (GALV) envelope proteins This technique improves transduction efficiency of mature lymphocytes and possibly hematopoietic stem cells The vesicular stomatitis virus (VSV) envelope protein allows direct membrane fusion, circumventing the need for a specific cell surface receptor, but toxicity of the envelope protein to both producer cell lines and target cells hinders development of this approach Lentiviruses Recently, there has been an intensive focus on the development of vectors based on lentiviruses such as the human immunodeficiency virus (HIV)-1 or Certain characteristics of HIV may overcome some of the limitations of the MuLV vectors Pseudotyping of HIV-based vectors with VSV or amphotropic envelope proteins would allow transduction of hematopoietic progenitor and stem cells Use of the HIV envelope gene would allow specific transduction of CD4+ targets HIV and other lentiviruses transduce target cells without the need for cell division The mechanism for this property is not fully understood But, the dissection of the HIV genome and incorporation of the nuclear transport mechanism(s) into otherwise standard MuLV vectors for gene therapy has not been successful Beyond these HEMATOPOIETIC STEM AND PROGENITOR CELLS AS TARGETS FOR GENE THERAPY 137 efforts, there are obviously major safety concerns that preclude clinical applications of HIV Absolutely convincing preclinical data regarding efficacy and lack of replication-competent virus must be obtained prior to human use Non-HIV-1 lentiviral vectors are also of great interest and are very early in development, as are vectors based on the human foamy virus (HFV), another retrovirus that appears to have little pathogenicity For adenoassociated virus (AAV), utility in hematopoietic stem cell gene transfer is unlikely However, applications requiring only transient expression in lymphocytes or dendritic cells are attractive Most recently, promising data has been obtained using AAV to transduce muscle cells in vivo, allowing prolonged production of soluble factors important in hematologic diseases such as factor IX for hemophilia or erythropoietin for anemia of chronic renal failure AAV vectors package 5.2 kb of new genetic material precluding the transfer of large genes such as factor VIII Adenovirus (Ad) vectors have been explored primarily for in vivo gene delivery for the transfection of both dividing and nondividing cells The immune response induced by Ad vectors, although a major disadvantage, is also being considered as a possible advantage for transduction of tumor cells with cytokines, co-stimulatory molecules, or other immune modulators in cancer vaccine protocols (see Chapter 13) These applications, thoroughly investigated in solid tumor animal models, are also being applied to hematologic malignancies such as leukemias and lymphomas Normal primitive hematopoietic cells can be transduced by Ad, but only with very highly concentrated vector preparations that also result in significant toxicity Transient expression in primitive cells may be of interest in manipulating homing after transplantation The simplest approach to gene transfer is to use naked plasmid deoxyribonucleic acid (DNA), with necessary control sequences and the transgene, as the vector The advantages of nonviral vectors include the lack of any risk of generation of replication-competent infectious particles, independence from target cell cycling during transduction, and elimination of antivector immune response induced by viral proteins There are few size constraints However, transduction efficiency of primary cells is very low, and physical methods such as electroporation or chemical shock used to increase gene transfer efficiency of plasmids into cell lines are either inefficient or toxic Encapsulation by lipsomes has been useful for some primary cell types, as has conjugation to molecular conjugates including polyamines and inactivated adenovirus However, none of these nonviral methods has shown any promise in the transduction of hematopoietic stem or progenitor cells Limited success has been reported transducing primary human lymphocytes with a device called the “gene gun,” introducing plasmid DNA into cells using colloid gold particles None of these vectors integrate, and expression levels are generally lower than reported with viral vectors HEMATOPOIETIC STEM AND PROGENITOR CELLS AS TARGETS FOR GENE THERAPY The concept of genetic correction or modification of HSCs has been an ongoing primary focus of gene therapy research The properties of both self-renewal and differentiation of HSC can provide for the continuous maintenance of the transgene in cells of hematopoetic origin, including red blood cells, platelets, neutrophils, and 138 GENE THERAPY FOR HEMATOLOGICAL DISORDERS lymphocytes Less obvious are the application to tissue macrophages, dendritic cells, and central nervous system microglial cells (Chapter 9) Lineage-specific control elements need to be included to allow for differential expression in the appropriate mature cell type; for example, the use of hemoglobin gene enhancers to target expression to red blood cells The genetic correction of these cells offer a potential curative, one-time therapy for a wide variety of congenital disorders such as hemoglobinopathies, immunodeficiencies, or metabolic storage diseases Gene therapy also allows consideration of novel approaches to malignancies and HIV infection such as differential chemoprotection and intracellular immunization (see Chapter 11) The feasibility of harvesting transplantable stem cells from the bone marrow (BM) and the maintenance in short-term ex vivo cell culture were a crucial advantages in early animal studies The discovery and isolation of hematopoietic cytokines in the mid-1980s allowed successful ex vivo culture and transduction, resulting in the first successful demonstration of efficient gene transfer into murine repopulating stem cells More recently, the discovery of alternative sources of stem cells such as mobilized PB and umbilical cord blood (UCB) broadens the potential for HSC gene therapy to neonates or conditions requiring very high dose stem cell reinfusion However, several obstacles have limited progress toward efficient gene transfer into HSCs Some are methodologic No in vitro assays exist to identify and quantitate true human stem cells Further, gene transfer strategies efficient in transduction of in vitro surrogates, such as day 14 colony forming units (CFU) or the primitive multipotential long-term culture initiating cells (LTCIC), have not resulted in similar high levels of transduction of actual repopulating cells in early clinical trials or large animal models Thus, optimization of protocols and testing of new approaches has been hampered An additional obstacle is the observation that the most primitive pluripotent hematopoietic cells appear to be predominantly in the quiescent G0 phase of the cell cycle These cells are thus resistant to transduction with MuLV retroviral vectors (Fig 6.2) Attempts to increase cycling of primitive cells during transduction by prolonged culture in the presence of various combinations of hematopoietic cytokines has resulted in decreased engrafting ability This is due to either loss of self-renewal properties, induction of apoptosis, or alteration in homing ability Additionally, a characteristic of primative hematopoietic stem and progenitor cells that inhibits efficient gene transfer is the low level of expression of receptors for a number of vectors including retroviruses and adenoassociated viruses Lastly, many clinical applications are in nonmalignant disease where the use of high-dose ablative conditioning therapy prior to reinfusion of genetically corrected autologous stem cells is unacceptably toxic Only with the use of high doses of stem cells can significant levels of engraftment occur without the use of high-dose conditioning chemotherapy or total body irradiation Preclinical Studies Initial retroviral gene transfer into murine hematopoietic repopulating cells was achieved in 1984 The discovery, availability, and application of various hematopoietic growth factors improved the efficiency of ex vivo retroviral transduction of murine hematopoietic cells Several different combinations of growth factors have been successfully used for supporting gene transfer into murine stem cells These HEMATOPOIETIC STEM AND PROGENITOR CELLS AS TARGETS FOR GENE THERAPY 139 include the combination of interleukin (IL-3), interleukin (IL-6), and stem cell factor (SCF) Inclusion of recently discovered early acting growth factors such as flt-3 ligand and megakaryocyte growth and development factor (MGDF)/thrombopoietin (TPO) have augmented the level of genetically modified cells These cytokines and growth factors maintain primitive cell physiology ex vivo and potentially stimulate primitive cells to cycle without differentiation They may also upregulate retroviral cell surface receptors Other manipulations that have been found beneficial in the murine system include (1) treatment of animals with 5-fluorouracil before marrow harvest to stimulate cycling of primitive cells, (2) the co-culture of target cells directly on a layer of retroviral producer cells or other stromal support, (3) the use of high titer (greater than 105 viral particles per ml) vector and (4) colocalization of vector and target cells using fibronectin-coated dishes Under these enhanced conditions, retroviral gene transfer into murine BM hematopoietic cells is now achieved in vivo with long-term marking at 10 to 100% in all cell lineages The persistence of vector sequences in short-lived granulocytes and in multiple-lineage hematopoietic cells from serially transplanted mice indicates that murine repopulating stem cells can be successfully modified with retroviral vectors Other supportive data include retroviral integration site analysis documenting the common transduced clones from different lineages The repopulation of murine stem cells in nonablative or partially ablative conditioning transplant models has been increased by pretreatment of recipient mice with G-CSF/SCF These results in the murine model have raised concerns about long-term expression of transgenes from integrated vectors Studies have shown poor or decreasing in vivo expression of the transgene or transgenes, especially with serial transplants, despite persistence of vector sequences A hypothesis for this down-regulation in expression is the methylation of specific sequences in the vector promoter and enhancer regions To counter this down-regulation in gene expression, many modifications have been made in basic MuLV vectors These include the exchange of control sequences in the long terminal repeats (LTRs) with sequences from other retroviruses with lineage specificity of expression and the mutagenesis of putative negative regulatory sequences Data suggest that modified vectors show improved long-term in vivo expression, although, equivalent long-term expression from standard MuLV vectors has been acheived under certain circumstances Evaluation of ex vivo gene transfer protocols using human cells mainly relies on in vitro progenitor cell assays, including CFU (representing committed progenitors), and long-term culture initiating cell (LTCIC), a putative in vitro stem cell surrogate Using similar optimized conditions to the murine model, 50% or more progenitor colonies were transduced by retroviral vectors Equally high LTCIC transduction has also been observed Although BM has been the traditional source for HSCs, optimized gene transfer into CFU or LTCIC indicates that mobilized PB and UCB can be sources for HSCs Purification for primitive cells by panning—the exposure of whole BM or mobilized PB to antibodies directed against cell surface antigens found only on primitive cells, such as CD34—followed by flow cytometric sorting or immunoabsorption results in the isolation of approximately to 5% of total cells These enriched progenitor cells have reconstituting properties in clinical transplantation protocols Selection for CD34+/CD38- or HLA-DR populations can further purify stem cells Recent studies show that CD34- cell populations also possess repopulating activity, 140 GENE THERAPY FOR HEMATOLOGICAL DISORDERS potentially arguing against the use of CD34-enriched cells for gene transfer and other applications Use of purified target cells permits practical culture volumes and higher vector particle to target cell ratios (MOI) during transduction, thereby increasing gene transfer efficiency As data emerge suggesting that the use of in vitro surrogate assays not predict levels of gene transfer seen in vivo in early human clinical trials, attention has refocused on studying in vivo repopulating cells One approach is the use of large animal models since the stem cell dynamics, cytokine responsiveness, and retroviral receptor properties appear to be similar between humans and nonhuman primates However, very few research centers have the facilities and resources to carry out such transplant studies, and thus current studies are feasible as small proof of principle experiments, with little ability to study the impact of changing multiple variables Rhesus or cynamologous monkeys and baboons are currently used most extensively The persistence of vector sequences was first observed in a rhesus monkey transplantation model in 1989 In this seminal study, the CD34-enriched marrow cells were transduced with a high titer vector producer cell line (greater than 108–10 viral particles per ml) secreting both human IL-6 and gibbon IL-3 However, this high titer producer cell line also produced significant titers of replication-competent helper virus due to recombination between vector and helper sequences in the producer cell line Thus, in vivo marking in these animals could not be interpreted Moreover, high-grade T-cell lymphomas were found in some recipients several months posttransplantation because of insertional mutagenesis by the replication-competent contaminating virus This complication resulted in wide agreement that it is absolutely necessary to use helper-free producer cell lines and vector stocks in any clinical application As well, it is necessary to assess safety in large animals before human clinical use Subsequent studies have documented long-term genetic modification of multiple hematopoietic lineages in primates using a number of different helper-free retroviral vectors These successful transductions have been performed in the presence of growth factors, using unpurified or CD34-enriched BM or mobilized PB cells Lower levels of gene-modified circulating cells were reported when compared to the mouse model (generally less than 0.01 to 1%), although similar optimized transduction conditions were used in both systems Improved marking levels of up to to 4% have been reported by transducing growth factor-stimulated PB or BM hematopoietic cells in the presence of a cell line engineered to express a transmembrane form of human SCF Recently, studies report further encouraging data when flt-3 ligand is added to the transduction cytokine combination, either in the presence of a fibronectin support surface or autologous stroma Marking levels of 10 to 20% in vivo for at least 20 weeks were confirmed by Southern blotting Some important results of retroviral transduction were obtained from the canine autologous transplantation model For instance, effective transduction of G-CSFmobilized peripheral blood repopulating cells was first observed in the dog Partially or fully ablative conditioning was necessary to obtain detectable engraftment with transduced HSCs Using this model, high levels (up to 10%) of transduced marrow CFU after transplantation have been reported using a 3-week long-term marrow culture for transduction and reinfusion without conditioning The expense and difficulty of transplanting large animals have resulted in the transplantation of gene-modified human hematopoietic cells in immunodeficient mice as an alternative model The major obstacle of this method is the low-level HEMATOPOIETIC STEM AND PROGENITOR CELLS AS TARGETS FOR GENE THERAPY 141 engraftment with human cells Improved results have been obtained by inclusion of co-transplantation of stromal cells secreting human IL-3, the use of more immunodeficient strains such as NOD/SCID, and transplantation into immunodeficient transgenic mice expressing human cytokines Identical retroviral integration sites were documented in human myeloid and T-cell clones obtained from a mouse posttransplantation, suggesting that pluripotent human HSCs were transduced Cord blood cells engraft with greater efficiency than adult BM or mobilized PB Thus studies have employ CB to a greater extent and extrapolate the data to other cell sources for gene therapy The predictive value of data derived from xenograft models remains to be proven through the direct comparison with results from human clinical studies, thereby tracking the same gene-modified cell population in both patients and immunodeficient mice Clinical Genetic Marking Studies Genetic marking of cells with an integrating vector is a unique method for tracking autologous transplanted cells and their progeny in vivo Early human clinical gene transfer trials used retroviral vectors carrying nontherapeutic marker genes to transduce a fraction of an autologous graft in patients undergoing autologous transplantation for an underlying malignancy These studies provided proof of principle and safety data Several studies used retroviral marking to track whether reinfused tumor cells contribute to relapse after autologous transplantation In two pediatric genemarking studies, unpurged autologous marrow from children with acute myeloid leukemia or neuroblastoma was briefly exposed to a retroviral vector carrying the Neo gene Genetically marked tumor cells were detected in several patients at relapse This observation suggested that the reinfused marrow had contributed to progression and that purging was necessary One adult marking study did not detect marked tumor cells in patients with acute leukemia at relapse, but overall transduction efficiencies in this study were lower Marked relapses were demonstrated in chronic myelogenous leukemia: bcr/abl+ marrow CFU-C were shown to contain the marker gene No marked relapses have been detected in adult patients with multiple myeloma and breast cancer transplanted with genetically marked bone marrow and peripheral blood cells However, the marrow and blood cells were CD34enriched before transduction, thus purging the starting population by at least logs of tumor cells Another outcome of these marking studies was to assess in vivo gene transfer efficiency In the pediatric study, a fraction of the bone marrow graft was briefly exposed to retroviral supernatant without growth factors or autologous stroma As many as to 20% of marrow CFU were shown to be neomycin-resistant between and 18 months posttransplantation, suggesting effective transduction and ongoing transgene expression This surprisingly high level of stable marked marrow progenitors may be explained in part by active cell cycle kinetics of the primitive HSCs from these children likely due to their young age Additionally, the primitive HSCs may have been undergoing hematopoietic recovery from high-dose chemotherapy just before BM collection However, only 0.1 to 1% of circulating mature cells were marked Treated adults have undergone autologous bone marrow and mobilized peripheral blood stem cell transplantation for multiple myeloma and breast cancer Bone 142 GENE THERAPY FOR HEMATOLOGICAL DISORDERS marrow and peripheral blood CD34-enriched cells were transduced with different retroviral vectors containing the Neo gene in order to assess the relative contribution to marking and engraftment of marrow and peripheral blood populations Transduction was performed for days in the presence of the cytokines IL-3, IL-6, and SCF Circulating marked cells were detected after engraftment in all patients Marked cells were also detected in three of nine recipients for over 18 months Although granulocytes, B cells, and T cells were positive for the transgene, the gene transfer efficiency was lower than in the pediatric studies Less than 0.1% of circulating cells were marked long term, and no high-level marking of marrow CFU-C was detected Because both the bone marrow and peripheral blood grafts contributed to long-term marking, this study documented that mobilized peripheral blood grafts can produce multilineage engraftment This study was also important evidence that allogeneic transplantation could be performed safely with this cell source These investigators also tested the brief single transduction protocol that was effective in the pediatric study, but no persistent marking was detected in adult patients Clinical Studies Using Therapeutic Genes A main objective of gene therapy is the replacement of defective or missing genes in congenital diseases A number of single-gene disorders such as the hemoglobinopathies, Fanconi anemia, chronic granulomatous disease, and Gaucher disease have been the focus of clinical trials The hematological deficiencies in these disorders can be successfully treated by allogeneic BMT, implying that normal stem cells can reverse the pathophysiology of the disorders Despite the low level of gene transfer into long-term repopulating stem cells achieved in large animal models and early human marking studies, several clinical trials exploring potentially therapeutic genes have been reported or are ongoing (Table 6.2) Important information has been obtained on safety and feasibility of stem cell engraftment without ablation, and there are glimmers of hope regarding clinical benefit Severe combined immunodeficiency due to adenosine deaminase (ADA) mutations was the first disease involving gene therapy of hematopoietic cells for several reasons The human ADA gene was cloned in the early 1980s and the small 1.5-kb (cDNA) could easily fit into a retroviral vector along a selectable marker gene such as Neo Even a low level of gene transfer efficiency might be efficacious because ADA normal cells should have an in vivo survival and proliferative advantage Thus, the correction of only to 5% of target cells may have clinical benefit Hematopoietic stem cells could be better gene correction targets than T cells in this and other immunodeficiency disorders because of the potential for permanent and complete reconstitution of the T-cell repertoire However, it has been difficult to achieve stable long-term efficient transduction of HSCs, thus T cells were the initial targets chosen To directly address this issue, two ADA-deficient children in Italy received both autologous bone marrow and mature T lymphocytes transduced with distinguishable retroviral vectors carrying both the ADA and Neo genes The patients were then repeatedly reinfused with both cell products without conditioning In the first year, vector-containing T cells originated from the transduced mature T cells; but, with time, there was a shift to vector-containing T cells originating from transduced bone marrow cells A normalization of the immune repertoire and Neo ADA, Neo Neo FACC BM BM and PB CD34+ cells UCB CD34+ cells EBV-specific cytotoxic lymphocytes T lymphocytes T lymphocytes BM BM PB CD34+ cells Chronic myeloid leukemia Breast cancer/multiple myeloma Severe combined immunodeficiency EBV-induced lymphoproliferative disorders (EBV-LPD) after BMT Severe combined immunodeficiency Severe combined immunodeficiency Acute leukemia Fanconi anemia HSV-TK, Neo, NGFR MDR1 PB CD34 cells Donor lymphocytes BM/PB CD34+ cells EBV-LPD, relapsed leukemia and GVHD after BMT Breast/ovarian/brain tumor p47 phox ADA, Neo ADA, Neo Neo Neo Chronic granulomatous disease + Neo BM Neuroblastoma Neo BM Acute leukemia Neo Gene Tumor infiltrating lyphocytes Target Cell Published Clinical Trials of Gene Transfer into Hematopoietic Cells Melanoma Disease TABLE 6.2 Transient or low-level gene transfer, no clear in vivo selection with chemotherapy Anti-EBV effect preserved, then elimination of GVHD by ganciclovir Prolonged (6 months) production of gene-corrected granulocytes (0.004–0.05%) Marking but no in vivo selection No marked tumor cells or persistence of marked hematopoietic cells Gene-corrected T cells from both transduced lymphocytes and stem cells Persistence of gene-corrected T cells (1–30%) Transient detection of marked T cells, then in vivo expansion with EBV activation Gene-marked T cells, and low-level marking of other lineages Persistence of marked cells of multiple lineages from PB and BM grafts Marked bcr/abl + CFU Marked normal CFU Marked tumor at relapse Persistence of marked normal CFU Marked tumor at relapse Persistence of marked normal CFU Detection of marked TILs in tumor Results HEMATOPOIETIC STEM AND PROGENITOR CELLS AS TARGETS FOR GENE THERAPY 143 144 GENE THERAPY FOR HEMATOLOGICAL DISORDERS restoration of cellular and humoral immunity were documented after gene therapy Data showed a surprisingly high number of marrow CFU resistant to neomycin This was despite the lack of conditioning and the authors hypothesize an in vivo selective advantage for gene-corrected cells of all lineages In genetic disorders diagnosed in utero, an exciting alternative approach is the use of cord blood These cells may contain relatively greater numbers of primitive repopulating cells more susceptible to retroviral transduction Moreover, early treatment is crucial before disease progresses chronically to irreversible damage The cord blood was collected at the time of delivery from three neonates diagnosed in utero with ADA deficiency The cells were CD34-enriched and transduced with an ADA/Neo retroviral vector The transformed cells were reinfused into the children without ablation Vector sequences were detected in circulating mononuclear cells and in granulocytes of all three children for longer than 18 months but at low levels of less than 0.05% However, when treatment with exogenous PEG-ADA was discontinued in one child, the proportion of vector-containing T cells increased to 10% or more This was an unexpected finding that implied in vivo selection for corrected cells Over time, however, the child’s immune function declined and PEGADA therapy restarted What can be concluded form the study is that the level of expression of ADA from the MuLV vectors remained low in unstimulated T cell in vivo These cells were not fully functional despite a possible survival advantage in the development of the T cells from precursors Fanconi anemia (FA) is a hematopoietic genetic disorder that may be an excellent candidate for gene therapy FA is a bone marrow failure syndrome, characterized by physical anomalies, and an increased susceptibility to malignancies Cells from these patients are hypersensitive to DNA-damaging agents FA can be functionally divided into at least five different complementation groups termed (A–E) Two different FA genes, FAC and FAA, have been identified from two different subsets of patients Phenotypic correction of these abnormalities in cells from two patient groups was successful after transduction with retroviral vectors carrying the FAC or FAA gene A possible in vivo survival advantage for gene-corrected primitive cells and their progeny has made FA an attractive candidate disease for stem cell gene therapy A clinical trial has tested this hypothesis using G-CSF-mobilized peripheral blood CD34+ cells from three FAC patients as targets The results of this trial suggest that gene complementation has at least transient positive effects on FA hematopoiesis as measured by progenitor growth and marrow cellularity However, no clear clinical benefit or in vivo survival advantage for transduced cells has been demonstrated Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency disorder of the NAPDH oxidase system and consequently of phagocytic cell function It is characterized by recurrent bacterial and fungal infections that induce granuloma formation and threaten the life of patient Four different genetic defects have been found to be responsible for this disease Current clinical management of CGD patients includes administration of antibiotics, interferon-g, or allogeneic BMT, but unsatisfactory clinical results make the development of gene therapy strategies highly desirable Low levels of correction may have clinical impact as healthy Xlinked CGD carrier females have been identified with only to 10% of normal levels of NADPH function In a clinical trial, five CGD patients with p47phox deficient have been reinfused with CD34+ peripheral blood stem cells transduced with LYMPHOCYTE GENE TRANSFER 145 a retroviral vector containing p47phox without conditioning Genetically-modified granulocytes were detected by PCR and correction of neutrophil oxidase activity was documented during the first few months after infusion But within months these cells became undetectable Similar results have been reported for a clinical trial carried out in patients with Gaucher disease Without ablation, vectorcontaining cells were detected at low levels and only transiently after reinfusion LYMPHOCYTE GENE TRANSFER Lymphocytes have characteristics that are advantageous for some gene therapy applications as compared to hematopoietic stem cells Lymphocytes are easily harvested in large numbers and can be cultured ex vivo without major perturbation of phenotype, immune responsiveness, or proliferative potential Lymphocytes may be repeatedly harvested and ablative conditioning is not necessary for persistence of infused cells Both preclinical animal data and early clinical trials have reported encouraging results However, they have also provided troublesome evidence of strong immune responses developing against exogenous genes expressed by lymphocytes Preclinical Studies Stable, long-term ex vivo expression of transgenes has been achieved by using a retroviral vector containing Neo and human ADA genes in both murine and human T lymphocytes Transduced murine lymphocytes could be selected by growth in G418 and subsequently expanded without changing their antigenic specificity Infusion of these cells into nude mice has resulted in the persistence of Neo-resistant cells that continued to produce human ADA for several months Modified transduction protocols have been explored to further improve gene transfer to lymphocytes Pseudotyping of MuLV vectors with a GALV envelope has increased lymphocyte transduction efficiency because lymphocytes appear to have more GALV receptors than amphotropic receptors Other technical improvements during transduction have included centrifugation to increase the interaction between target cell and virus, phosphate depletion to up-regulate the amphotropic or GALV receptors, and low-temperature incubation to stabilize vector particles Under these optimized conditions, up to 50% of lymphocytes can be transduced ex vivo without changes in viability, phenotype, or expansion capability In an in vivo marking study, rhesus peripheral blood lymphocytes were transduced successfully with a vector encoding the Neo gene and HIV-1 tat/rev antisense sequences using these techniques Following reinfusion, to 30% of circulating CD4+ cells contained the vector for at least several months, and lymph node sampling demonstrated that these cells could traffic normally Clinical Genetic Marking Studies The initial controlled and monitored human gene transfer study used retroviral marking to monitor the fate of tumor-infiltrating lymphocytes (TIL) in vivo Lowlevel marking was detected in tumor deposits However, marking levels were too 146 GENE THERAPY FOR HEMATOLOGICAL DISORDERS low to assess any preferential trafficking of TIL cells to residual tumor In subsequent gene marking studies, behavior of transduced donor lymphocytes was studied in patients undergoing allogeneic transplantation To control Epstein–Barr virus (EBV)-induced lymphoproliferative disorders (EBV-LPD) postallogeneic BMT, EBV-specific donor T cells were isolated, expanded, and gene marked in culture with EBV-transformed donor lymphoblasts as stimulators After transplantation, the transduced T cells were reinfused, and two to three orders of magnitude expansion of marked cells were measured in vivo EBV-specific cytotoxity in the peripheral blood was greatly enhanced after the infusions Although circulating marked cells became undetectable by to months after infusion, the persistence of memory cells from the infusion product was inferred in a patient with detectable marked lymphocytes in the blood after reactivation of latent EBV Suicide Gene Transfer A similar approach has been utilized in patients with EBV-LPD with the modification of incorporating the herpes simplex virus thymidine kinase (HSV-tk) gene into the retroviral vector This suicide gene converts the nontoxic prodrug ganciclovir to a toxic metabolite that kills the tk-expressing cell by inhibition of DNA synthesis The inclusion of this gene in vectors allows elimination of transduced cells in vivo simply by ganciclovir administration postinfusion For example, ganciclovir treatment could abrogate graft-versus-host disease (GVHD) in allogeneic BMT recipients if most of the allogeneic T cells contain the tk gene This strategy depends on inclusion of a cell surface marker gene in the vector to allow positive selection of transduced cells before reinfusion This would allow almost all infused cells to contain the tk gene and thus be sensitive to ganciclovir killing In allogeneic transplantation, donor lymphocytes play a therapeutic role in both graft-versus-leukemia (GVL) and immune reconstitution However, their application is limited by the risk of severe GVHD In a clinical trial, eight patients who relapsed or developed EBV-induced lymphoma after T-depleted BMT were treated with donor lymphocytes transduced with HSV-tk suicide gene The transduced lymphocytes survived for up to 12 months, resulting in antitumor activity in five patients Three patients developed GVHD, which could be effectively controlled by ganciclovir-induced elimination of the transduced cells This study and other studies where patients with HIV disease received ex vivo expanded autologous lymphocytes transduced with a tk-hygromycin-resistant vector have reported troublesome evidence of an immune response developing against foreign gene products, such as herpes tk or drug-resistant genes This immune response limits the persistence of transduced cells, as well as repeated infusions Therapeutic Genes As noted earlier, the initial human gene therapy study used T lymphocytes as targets Two children with severe combined immunodeficiency due to ADA deficiency received multiple infusions of autologous T cells transduced with a retroviral vector containing the human ADA gene Both patients showed relative improvements in circulating T numbers and cellular and humoral immunity In one child, the T-cell numbers rose to normal, lymphocyte ADA levels increased to CURRENT PROBLEMS AND FUTURE DIRECTIONS 147 roughly half that seen in heterozygote carriers of the disease, and the vector was detected in peripheral T lymphocytes at a concentration of approximately copy per cell In the second child, the T cell level rose temporarily during the infusions and then fell back T-cell ADA activity did not increase, and only 0.1 to 1% of circulating T cells contained the vector even after multiple infusions Both patients showed persistence of vector-containing cells for more than years after the last Tcell infusion, which shows that transfused peripheral T cells can have a long life span The expression level of ADA in these lymphocytes appears to be low, becoming significant with ex vivo activation Thus, vector modifications may be needed to improve expression Internationally, a similar study has been performed in one patient and the percentage of peripheral blood lymphocytes carrying the transduced ADA gene has remained stable at 10 to 20% during the 12 months since the fourth infusion ADA enzyme activity in the patient’s circulating T cells, which was only marginally detected before gene transfer, increased to levels comparable to those of a heterozygous carrier individual This level was associated with increased Tlymphocyte counts and improvement of immune function CURRENT PROBLEMS AND FUTURE DIRECTIONS In Vivo or Ex Vivo Selection The observed low efficiency of gene transfer into hematopoietic stem and progenitor cells or other targets transduced ex vivo may be compensated by either positive selection of transduced cells before reinfusion or in vivo after engraftment Rapid selection of transduced cells can be carried out using marker genes encoding proteins detectable by fluorescence-activated cell sorting (FACS) or other immunoselection techniques The human cell surface protein CD24 or its murine analog, heat-stable antigen (HSA), has been tested as a selectable marker Both small proteins (200 to 250 bp) take up little space in vector constructs, and noncrossreacting antibodies are available Murine cells transduced with a vector containing human CD24 and selected before transplantation result in long-term reconstitution with a very high proportion of cells containing the vector A vector expressing HSA allowed enrichment for transduced human progenitor cells However, CD24 and HSA are glycosylphophatidylinositol-linked surface proteins This class of proteins has been shown to be transferred from cell to cell both in vitro and in vivo, possibly complicating interpretation A truncated, nonfunctional form of the human nerve growth factor receptor (NGFR) has also been developed as a selectable marker for hematopoietic cells, because hematopoietic cells not express endogenous NGFR Preclinical studies and early clinical trials have shown that transduction and sorting of lymphocytes using this marker is sensitive and specific However, the introduction of new cell surface proteins has the theoretical disadvantage of altering trafficking or cell/cell interactions upon infusion of transduced cells Alternative cytoplasmic markers such as jellyfish green fluorescent protein (GFP) are naturally fluorescent, avoiding the need for antibody staining Reconstitution with enriched GFP+ cells and long-term expression of GFP in multiple bonemarrow-derived cell lineages have been achieved in the murine model However, a large animal study demonstrated that CD34-positive GFP-positive progenitor cells 148 GENE THERAPY FOR HEMATOLOGICAL DISORDERS selected after 5-day culture in the presence of multiple cytokines are able to produce mature CD13+ cells in the short-term But these cells failed to engraft in the medium to long term In human studies, the positive selection of transduced lymphocytes using selection markers has already been achieved The further expansion of transduced cells shows no changes in phenotype or in vivo function However, it is still difficult to use ex vivo selection strategies on human hematopoietic stem cells posttransduction due to a low gene transfer efficiency The major concern is that too few stem cells remain to allow safe and rapid hematopoietic reconstitution after enrichment of transduced cells, especially if ablative conditioning will be used A potential solution to this problem is ex vivo expansion of selected transduced cells before reinfusion It is unknown whether true long-term repopulating cells can be expanded or even maintained ex vivo using current culture conditions Expanded cells have been documented to engraft lethally irradiated or stem-cell-deficient mice However, a competitive disadvantage of ex vivo cultured cells against endogenous stem cells was shown in a nonablative model In the ablative rhesus model, transduced CD34+ cells expanded for 10 to 14 days ex vivo competed poorly against cells transduced and cultured for days, despite to log expansion of total cells and CFU In vivo-selectable drug-resistant genes have been incorporated into retroviral vectors There are at least two possible applications for this in vivo drug selection strategy: (1) induction of chemoprotection and (2) in vivo positive selection of genetically modified cells Bone marrow suppression is one of the most common toxicities of chemotherapy regimens One approach to increase the tolerated dose of chemotherapy is to introduce the human multidrug resistance (MDR1) gene into bone marrow stem and progenitor cells The protein product of this gene, called P-glycoprotein, can extrude many chemotherapy drugs out of cells, thereby resulting in a drug-resistant phenotype These drugs include the anthracyclines, taxol, vinca alkaloids, and epipodophyllotoxins Another potential application is to incorporate the gene into a vector with another gene of interest (e.g., glucocerebrosidase) to allow in vivo enrichment of the percentage of gene-modified cells into a therapeutically beneficial range by administration of MDR-effluxed drugs Mice engrafted with MDR1-transduced marrow cells tolerate higher dose of MDReffluxed drugs, and develop increasing percentages of circulating vector-containing cells These cells are stable without further treatment suggesting selection at an early stem or progenitor cell level The human clinical trials piloting this marrowprotective approach have been performed in patients undergoing autologous BMT for solid tumors such as ovarian and breast cancer No clear evidence of chemoprotection or in vivo selection has been obtained However, transductions in these trials were used in suboptimal protocols, and the level of marking was extremely low or undetectable Thus, the results are not surprising There has been a recent report of an aggressive myeloproliferative and eventually leukemic syndrome occurring in mice transplanted with MDR1-transduced marrow cells that were expanded ex vivo This poor outcome possibly implicates the MDR1 gene product in leukemogenesis and may terminate future clinical applications of MDR1 Other drug-resistant genes have also been studied in vitro and in murine models These include 06-alkylguanine-DNA-alkyltransferase or glutathione S-transferase, which confer protection against alkylating agents, and mutant dihydrofolate reduc- CURRENT PROBLEMS AND FUTURE DIRECTIONS 149 tases (DHFRs) that confer resistance to trimetrexate as well as other antimetabolites Each is very promising and may reach clinical trials in the near future Issues with these strategies for chemoprotection are that nonhematologic toxicity may rapidly become limiting, and patients will not be protected from those side effects by engraftment with gene-modified, protected stem cells Alternative Vectors The limitations of retroviral vectors has led to an intensive search for other viral vectors that can both transduce quiescent cells and integrate permanently into their genome One type of candidate are vectors based on HIV HIV vectors can transduce a high percentage of CD34+ hematopoietic cells In addition, G0/G1 primitive hematopoietic cells engrafting NOD/SCID mice can be transduced by lentiviralbased vectors and maintain their primitive phenotype, pluripotentiality, and transgene expression Although AAV has been investigated extensively for hematopoietic cell gene transfer, most current data argues against the use of AAV for these applications This is because of inefficient AAV vector integration Several laboratories have reported high transduction efficiency of both human and murine hematopoietic progenitors, as assayed by PCR or G418-resistant CFU-C, but primate studies indicate no advantage over retroviral vectors in gene transfer into repopulating stem cells Gene Correction Current gene transfer strategies rely to a large extent on random insertion of a complete new copy of a defective gene or a corrective gene A new copy is inserted even if the defect in the original gene is only a point mutation Newer strategies aimed at repairing mutations in the endogenous gene are thus very attractive One novel strategy is the correction of a mutation in the b-globin gene in EBV-transformed lymphocytes derived from patients with sickle cell anemia by use of chimeric RNADNA oligonucleotides The analysis was only by PCR, with inherent potential for misinterpretation, and has not been reproduced However, if this approach, or other similar methods, can reproducibly correct mutations in nondividing human hematopoietic stem cells, it will revolutionize the gene therapy field Immune Responses to Vectors and Transgenes Immune responses against vector proteins or transgene-encoded proteins are clearly an obstacle to successful gene therapy Repeated in vivo administration of complex vectors stimulates an active immune response to vector proteins This results in clearance of subsequent vector before in vivo transduction as well as causing damage to transduced tissues To overcome this problem, modified adenoviral vectors have been developed with minimal residual adenoviral genes Nonhuman marker genes such as the Neo gene or suicide genes such as tk gene included in vectors for selection may also induce an immune response The therapeutic gene itself may induce an immune response if the patient completely lacks the endogenous gene product 150 GENE THERAPY FOR HEMATOLOGICAL DISORDERS In a murine allogeneic skin transplantation model, foreign genes expressed by hematopoietic stem cells and their progeny induce immune tolerance across MHC barriers However, foreign transgene products expressed in lymphocytes, myocytes, and other non-stem cells clearly are capable of inducing an immune response A dual strategy of engraftment of transduced stem cells and actual transduced target cells that need to be corrected (i.e., lymphocytes or muscle cells) may be necessary to induce tolerance Immune rejection of transduced cells has been controlled partially with immunosuppression using agents such as cyclosporine, cyclophosphamide, or IL-12 But these pharmacologic approaches are not desirable or practical for most gene therapy applications for hematological disorders Thus, improved vector design and possible inclusion of anti-rejection mechanisms in the vector constructs are more desirable SUMMARY The first genetic disease elucidated at the molecular level was sickle cell anemia Gene therapy of disorders such as the hemoglobinopathies requires high-level correction and has been difficult to achieve But it seems likely that some hematological disorders, such as severe combined immunodeficiency caused by ADA deficiency or chronic granulomatous disease, will become amenable to effective gene therapy A better understanding of stem cell biology as well as the development of simple and reliable vectors are necessary for further progress The wide variety of novel approaches for gene transfer currently being developed are certain to eventually achieve the promise of gene therapy first envisioned a decade ago KEY CONCEPTS • • Hematopoietic stem cells (HSCs) have the ability to self-renew and differentiate into all lineages of the hematopoietic system, including the reticuloendothelial system and central nervous system microglial cells HSCs are easily collected from marrow, stimulated peripheral blood, and cord blood and can be cultured and transduced ex vivo before intravenous reinfusion These features have made HSCs an ideal target for gene therapy of a wide variety of congenital disorders (immunodeficiencies, hemoglobinopathies, metabolic storage diseases) and acquired diseases (HIV infection and malignancies) Gene transfer into HSCs has been hampered by several biological obstacles, and early clinical trials have not shown clinically relevant levels of gene transfer in most instances Problems include: (1) No in vitro assay to identify and quantitate true stem cell exists (2) HSCs appear to be predominantly in G0 phase of the cell cycle, making them resistant to proviral integration with the retroviral vectors in current clinical use (3) The receptors for a number of vectors including retroviruses and adenoassociated virus are expressed at low levels on HSCs (4) Chromosomal integration is necessary for delivery of the transgene to progeny cells Non-DNA integrating delivery systems such as ade- SUGGESTED READINGS • 151 noviruses will result in only transient expression and thus correction Improved gene transfer efficiency has been reported in many relevant preclinical studies, especially large animal models by the inclusion of new hematopoietic growth factors and fibronectin or stroma during transduction, pseudotyping of retroviral vectors, and application of lentiviral vectors Lymphocytes have several features that make them more attractive than HSCs as targets for gene therapy They are easily harvested, circulate in large numbers, and can be cultured ex vivo without changes of phenotype, immune responsiveness or proliferative potential They may be repeatedly harvested, and ablative conditioning is not necessary for persistence of infused cells They are used in the gene therapy for congenital and acquired immunodeficiencies, malignancies, and GVHD SUGGESTED READINGS Gene Transfer Kiem HP, Andrews RG, Morris J Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor Blood 92:1878–1886, 1998 Liu JM, Young NS, Walsh CE, et al Retroviral mediated gene transfer of the Fanconi anemia complimentation group C gene to hematopoietic progenitors of group C patients Hum Gene Therapy 8:1715–1730, 1997 Lutzko C, Dube ID, Steward AK Recent progress in gene transfer into hematopoietic stem cells Crit Rev Onol Hematol 30:143–158, 1999 Rosenberg SA, Aebersold P, Cornetta K Gene transfer into humans—immunotherapy of patients with advanced melanoma, using tumor-infiltrating lymphocytes modified by retroviral gene transduction N Engl J Med 323:570–578, 1990 Gene Marking Brenner MK, Rill DR, Holladay MS Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients Lancet 342:11347, 1993 Brenner MK, Rill DR, Moen RC Gene-marking to trace origin of relapse after autologous bone marrow transplantation Lancet 341:85–86, 1993 Deisseroth AB, Zu Z, Claxton D, et al Genetic marking shows that Ph+ cell present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow transplantation in CML Blood 83:3068–3076, 1994 Dunbar CE, Cottler-Fox M, O’Shaughnessy J, et al Retrovirally-marked CD34-enriched peripheral blood and bone marrow and cells contribute to long-term engraftment after autologous transplantation Blood 85:3048–3057, 1995 Tisdale JF, Hanazono Y, Sellers SE, et al Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability Blood 92:1131–1141, 1998 152 GENE THERAPY FOR HEMATOLOGICAL DISORDERS Gene Therapy Blaese RM, Culver KM, Miller AD, et al T lymphocyte-directed gene therapy for ADASCID: Initial trial results after years Science 270:475–480, 1995 Bordignon C, Notarangelo LD, Nobili N, et al Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients Science 270:470–475, 1995 Hesdorffer C, Ayello J, Ward M, et al Phase I trial of retroviral-mediated transfer of the human MDR1 gene as marrow chemoprotection in patients undergoing high-dose chemotherapy and autologous stem-cell transplantation J Clin Oncol 16:165–172, 1998 Heslop HE, Ng CYC, Li C, et al Long-term restoration of immunity against Epstein-Barr virus infection by adoptive transfer of gene-modified virus-specific T lymphocytes Nat Med 2:551–555, 1996 Kohn DB, Weinberg KI, Nolta JA, et al Engraftment of gene-modified umbilical cord blood cells in neonates with adenosine deaminase deficiency Nat Med 1:1017–1023, 1995 Malech HL, Maples PB, Whiting-Theobald N, et al Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease Proc Natl Acad Sci USA 94:12133–12138, 1997 Riddell SR, Elliott MM, Lewinsohn DA, et al T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients Nat Med 2:216–223, 1996 Rill DR, Santana VM, Roberts WM, et al Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells Blood 84:380–383, 1994 Rooney CM, Smith CA, Ng CYC, et al Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoproliferation Lancet 345:9–13, 1995 Hematopoietic Stem Cell Gene Therapy Dunbar CE Gene transfer to hematopoietic stem cells: Implications for gene therapy of human disease Annu Rev Med 47:11–20, 1996 Kume A, Hanazono Y, Mizukami H, Urabe M, Ozawa K Hematopoietic stem cell gene therapy: A current overview Int J Hematol 69:227–233, 1999  ... PROGENITOR CELLS AS TARGETS FOR GENE THERAPY 143 144 GENE THERAPY FOR HEMATOLOGICAL DISORDERS restoration of cellular and humoral immunity were documented after gene therapy Data showed a surprisingly... patient completely lacks the endogenous gene product 150 GENE THERAPY FOR HEMATOLOGICAL DISORDERS In a murine allogeneic skin transplantation model, foreign genes expressed by hematopoietic stem... of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability Blood 92:1131–1141, 1998 152 GENE THERAPY FOR HEMATOLOGICAL DISORDERS Gene Therapy